The process of transcription is essential for all living cells. Transcription is accomplished in four steps: promoter binding, RNA chain initiation, RNA transcript elongation, and RNA transcript termination. Regulation of transcription can occur at each of these steps. The long-term research goal of this proposal is to understand the mechanism and the regulation of transcript elongation by eukaryotic RNA polymerases in chromatin. When eukaryotic genes are activated for transcription, their chromatin structure changes to accommodate transcription factors and to allow efficient transcription by RNA polymerases. However, for many genes there is evidence that transcribed regions are covered with nucleosomes. This raises the questions: 1. How do polymerases transcribe through the chromatin barrier? 2. Can the barrier change the rate of transcript elongation? These questions will be addressed in a highly purified transcription system in vitro. We will analyze transcription of homogeneous and well-defined mono- and polynucleosomal chromatin templates using biochemical and molecular genetic techniques, focusing on analysis of eukaryotic RNA polymerase II. The specific aims are: 1. To ascertain the mechanism of transcription through chromatin by RNA polymerase II: the fate of the histone octamer and the nature of nucleosomal barrier to transcription. Information to be obtained here will provide a structural framework for understanding of transcription through chromatin and its rate-limiting step(s). 2. To ascertain the rote of elongation factors, modifications of chromatin structure, and modifications of RNA polymerase II in facilitating transcription through chromatin. These experiments will lead to better understanding of regulation of transcription at the level of transcript elongation. The discovery that some elongation factors play important roles in oncogenesis underscores the potential clinical significance of analysis of the mechanism of transcript elongation.